The invention is directed to a method which specifically amplifies a target nucleic acid region of unknown nucleotide sequence which is disposed downstream from (that is, in the 3xe2x80x2 direction from) a portion of the nucleic acid target region having a known nucleotide sequence.
The polymerase chain reaction (PCR) enables amplification of a nucleic acid sequence (i.e., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) which lies between two regions of known nucleotide sequence. See Mullis et al., U.S. Pat. Nos. 4,683,202 and 4,683,195, the teaching of which is incorporated herein by reference. Oligonucleotides complementary to the known 5xe2x80x2 and 3xe2x80x2 sequences flanking the unknown portion of the target nucleic acid serve as xe2x80x9cprimersxe2x80x9d in the PCR procedure. In the PCR, double-stranded target nucleic acid is first melted to separate the two strands. The oligonucleotide primers complementary to the known 5xe2x80x2 and 3xe2x80x2 portions of the segment which is desired to be amplified are then annealed to the target (or xe2x80x9ctemplatexe2x80x9d) nucleic acid. The portions of the nucleic acid target where the primers anneal serve as starting points for the synthesis of new complementary nucleic acid strands. This process utilizes an added DNA or RNA polymerase, most often Taq DNA polymerase, although other DNA polymerases are known. The enzymatic synthesis of the complementary nucleic acid strands is known as xe2x80x9cprimer extension.xe2x80x9d The orientation of the 5xe2x80x2 and 3xe2x80x2 primers with respect to one another is such that the 5xe2x80x2 to 3xe2x80x2 extension product from each primer contains, when extended far enough, the sequence which is complementary to the other primer. Thus, each newly synthesized nucleic acid strand becomes a template for synthesis of yet another nucleic acid strand beginning with the opposite primer. Repeated cycles of melting, annealing of primers, and primer extension lead to a (near) doubling of nucleic acid strands with each cycle. Each new strand contains the sequence of the target nucleic acid beginning with the sequence of the first primer and ending with the sequence of the second primer.
A key and inescapable requirement of the PCR is the need for two primers which are complementary to the 5xe2x80x2 and 3xe2x80x2 ends of the target nucleic acid which is to be amplified. Not only must the primers be made complementary to two known and non-contiguous portions of the target nucleic acid, the primers must be oriented such that their 3xe2x80x2 extension products proceed toward each other. If the sequences at both ends of the segment to be amplified are not known, complementary primers cannot be synthesized and the standard PCR protocol cannot be performed.
The object of the present invention is to overcome the need for sequence information at both ends of the segment to be amplified. In short, the present invention comprises a method which enables the PCR to be performed when sequence information is known for only a single region of the target Nucleic acid. The invention also provides a method for contiguously amplifying and sequencing a very long nucleic acid region without having to sub-clone the nucleic acid to arrive at nucleic acid targets of smaller size.
The invention is drawn to a method of specifically amplifying a selected region of nucleic acid (DNA or RNA). The method comprises first annealing a 5xe2x80x2-blocked oligonucleotide primer to a target nucleic acid molecule. The target nucleic acid molcule has a region of known nucleotide sequence and a region of unknown nucleotide sequence 3xe2x80x2 to the known nucleotide sequence. The sequence to be selectively amplified is the region of unknown nucleotide sequence. The oligonucleotide primer is designed to anneal to the region of known nucleotide sequence.
The 5xe2x80x2-blocked oligonucleotide primer, now annealed to the known region of the target nucleic acid, is then extended in the 3xe2x80x2 direction to yield a DNA strand complementary to at least a portion of the region of unknown nucleotide sequence in the target nucleic acid molecule.
The complementary nucleic acid strand so synthesized is then coupled at its 3xe2x80x2 terminus to a 3xe2x80x2-blocked oligonucleotide of known identity, thereby yielding a nucleic acid fragment of known sequence at its 5xe2x80x2 and 3xe2x80x2 termini. The nucleic acid fragment so formed, which has a known sequence at is 5xe2x80x2 and 3xe2x80x2 terminus is then amplified using conventional means, preferably using the PCR.
The primary advantage of the present invention is that it enables the specific amplification of long stretches of DNA or RNA of unknown nucleotide sequence when only a single region of the target DNA or RNA is of known sequence. More specifically, the invention enables specific PCR amplification of a region of DNA or RNA of unknown sequence which lies 3xe2x80x2 to a region of known sequence, using only a single primer of known sequence.
A host of other advantages become immediately apparent in view of the ability to amplify specifically target nucleic acid of unknown sequence using only a single primer. Most readily apparent is that the method can be used to amplify reiteratively a first (initially) unknown region of DNA or RNA, sequence the unknown region, and then use the newly-generated sequence information to amplify a second (initially) unknown region of DNA or RNA which is contiguous to the first region, and so on, ad infinitum.
This aspect of the invention is tremendously advantageous for sequencing very long nucleic acid molecules using the PCR procedure because it requires only limited sequence information for the target nucleic acid. Moreover, the information generated in each cycle of the process enables contiguous regions of nucleic acid of unknown sequence to be successively amplified (specifically) and then sequenced (or manipulated by any other means, such as by restriction digestion, cloning, etc.). Using the present invention, the starting knowledge one needs to possess is simply the nucleotide sequence of a single, very small region of target DNA or RNA. The method described herein can then be used to amplify, in continguous fashion, very long regions of DNA or RNA of unknown sequence, such as undigested genomic DNA, without the need for sub-cloning it into smaller fragments. By sequencing the amplified product, the process can by applied reiteratively to elucidate the sequence of vast stretches of previously unsequenced DNA or RNA.
Another advantage of the invention is that, like convetional PCR, it is readily automatable. Automatable oligonucleotide synthesizers are now widely available commercially, as are automatable PCR cycling blocks and automatable DNA sequencing machines. For example, such machines are available in the United States from Perkin-Elmer Corporation, Foster, Calif.; Beckman Instruments, Columbia, Md., and others. Because the process can be performed reiteratively, it is extremely well-suited to automation using existing equipment. After generating the first 5xe2x80x2-blocked primer, the annealing, initial amplifying, coupling of a 3xe2x80x2 terminus, and subsequent amplifying and sequencing of the target DNA are all automatable using existing equipment. Knowledge gained from each successive cycle can be used to generate (automatically) a new 5xe2x80x2-blocked primer for use in the next cycle.
Further aims, objects, and advantages of the amplification method will become apparent upon a complete reading of the Detailed Description which follows.